The present invention provides compositions and methods of modulating the expression of Her-1. Many human tumors have been found to overexpress this receptor and such enhanced expression has been shown to be correlated with poor prognosis. In particular, this invention relates to oligonucleotides specifically hybridizable with nucleic acids encoding human Her-1. These oligonucleotides have been found to inhibit the expression of Her-1.
Her-1, also known as epidermal growth factor receptor (EGFR), is a specific receptor for epidermal growth factor (EGF) and transforming growth factor-xcex1 (TGF-xcex1). When these mitogenic polypeptides bind to Her-1, tyrosine kinase activity of the receptor is induced, and this in turn triggers a series of events which regulate cell growth. A number of malignant and non-malignant disease conditions are now believed to be associated with Her-1, particularly aberrant expression of Her-1. Aberrant expression includes both increased expression of normal Her-1 and expression of mutant Her-1. Overexpression of Her-1 is found in many human tumors including most glioblastomas and breast, lung, ovarian, colorectal, bladder, pancreatic, squamous cell and renal carcinomas. Elevated Her-1 levels correlate with poor prognosis in human tumors. Her-1 is also implicated in nonmalignant diseases, such as psoriasis. The sequence of the mRNA encoding human Her-1 is known. Ullrich et al., Nature, 1984, 309, 418; GenBank Accession Number X00588; and Kraus et al., Proc. Natl. Acad. Sci. USA, 1989, 86, 9193; GenBank Accession Number M29366. The gene encoding Her-1 is also known as ERBB3 or c-erb-B1. Two Her-1 transcripts typically appear on Northern blots, one measuring 10 kb and one measuring 5.6 kb.
A number of inhibitors of Her-1 have been shown to be effective in inhibiting the growth of human tumor cells. Monoclonal antibodies to Her-1 and drugs which inhibit Her-1 tyrosine kinase activity can inhibit the growth of human cancer cell xenografts in nude mice. Normanno et al., Clin. Cancer Res., 1996, 2, 601. The drug PD153035, which inhibits Her-1 tyrosine kinase activity, can inhibit the growth of A431 cells in nude mice, and tyrphostins, which inhibit the activity of Her-1 as well as other tyrosine kinases, have been shown to inhibit the growth of squamous carcinoma in nude mice. Kunkel et al., Invest. New Drugs, 1996, 13, 295 and Yoneda et al., Cancer Res., 1991, 51, 4430.
Vectors expressing Her-1 nucleic acid sequences in an orientation complementary to mRNA have been used to study the effects of Her-1 on proliferation of cultured cancer cells. Transfectants of the human epidermoid carcinoma KB cell line expressing Her-1 cDNA or RNA sequences in an orientation complementary to mRNA exhibited restored serum-dependent growth and impaired colony formation and growth in agar. Moroni et al., J. Biol. Chem., 1992, 267, 2714. Human pancreatic carcinoma cells of the PC-7 cell line transfected with vectors expressing Her-1 cDNA sequences in an orientation complimentary to mRNA showed inhibited cell growth, colony formation and tumorigenicity in nude mice. Liu et al., Chinese Medical Journal., 1995, 108, 653. Transfection of human colon cancer cell lines with Her-1 RNA expression vectors producing an oligonucleotide complementary to mRNA caused a reduction in cell proliferation and ability to grow on soft agar. Rajagopal et al., Int. J. Cancer, 1995, 62, 661. Human rhabdomyosarcoma cells transfected with a plasmid expressing Her-1 cDNA in an orientation complementary to mRNA had greatly impaired proliferation. De Giovanni et al., Cancer Res., 1996, 56, 3898.
Considerable research is being directed to the application of oligonucleotides complementary to mRNA and other oligomers for therapeutic purposes. Oligonucleotides complementary to mRNA have already been employed as therapeutic moieties in the treatment of disease states in animals and man, and compositions comprising oligomers complementary to mRNA have been shown to be capable of modulating expression of genes implicated in viral, fungal and metabolic diseases. Further, oligonucleotides complementary to mRNA have been safely administered to humans and clinical trials of approximately a dozen oligonucleotide drugs targeted to viral and cellular gene products are underway.
Oligodeoxyribonucleotides complementary to mRNA targeted to Her-1 have been encapsulated into liposomes linked to folate via a polyethylene glycol linker and delivered into cultured human epidermoid carcinoma KB cells. The oligonucleotides were a phosphodiester (Pxe2x95x90O) 15-mer complementary to the Her-1 gene stop codon, or the same sequence with three phosphorothioate (Pxe2x95x90S) linkages at each end. Both of these oligonucleotides reduced KB cell proliferation by greater than 90% after treatment with 3 xcexcM oligonucleotide in folate-PEG-liposomes. In contrast, free Pxe2x95x90O oligonucleotide caused almost no growth inhibition, and free Pxe2x95x90S-capped oligonucleotide caused only a 15% growth inhibition, even at this high dosage level. Her-1 expression, measured by indirect immunofluorescence, was virtually abolished in cells treated with either of the folate-PEG-liposome-encapsulated oligonucleotides but Her-1 expression was qualitatively similar to untreated cells after treatment with free oligonucleotide. Wang et al., Proc. Natl. Acad. Sci. USA, 1995, 92, 3318.
A 15-mer phosphorothioate oligonucleotide complementary to the translation initiation region of Her-1 mRNA was found to inhibit cell proliferation by over 25% in A431 cells, derived from a vulval carcinoma. This activity, though dose-dependent from 1-25 xcexcM, was not mediated by an antisense mechanism, as demonstrated by a lack of reduction in either Her-1 protein or mRNA after oligonucleotide treatment. In addition, an 18-mer oligonucleotide complementary to mRNA targeted to the same region had no effect even at the highest (25 xcexcM) dose, and neither oligonucleotide had any effect in the two other tumor-derived cell lines tested. Coulson et al., Mol. Pharm., 1996, 50, 314.
The suppression of growth of pancreatic carcinoma cell lines by undisclosed oligonucleotides complementary to mRNA inhibiting the expression of TGF-xcex1 and/or the Her-1 has been reported. Hall et al., unpublished data, reported in Hall and Lemoine, Models of Pancreatic Cancer, in Cancer Surveys, Volume 16: The Molecular Pathology of Cancer, 1993, p. 135-155.
Rubenstein et al. have reported treatment of established human-derived prostate tumor xenografts in nude mice by intralesional injection of oligonucleotides complementary to mRNA directed against mRNAs encoding TGF-xcex1 and Her-1. The oligonucleotides included 39-mers complementary to 18 bases located 5xe2x80x2 and 3xe2x80x2 from the AUG mRNA translation initiation codon of either TGF-xcex1 or Her-1 sequence. The oligonucleotides were phosphorothioated at each of three terminal bases at both the 5xe2x80x2 and 3xe2x80x2 ends. The oligonucleotides were administered either alone or in combination, with the combination treatment proving most effective. J. Surg. Oncol., 1996, 62, 194. In U.S. Pat. No. 5,610,288, Rubenstein et al. disclose polynucleotides of about 20 to 50 nucleic acid bases, most preferably about 40 nucleic acid bases in length, which preferentially hybridize to the start codon of the mRNA encoding Her-1. A preferred embodiment is a 39-mer including 18 bases complementary to the 5xe2x80x2 side of the translation initiation codon. This oligonucleotide inhibited PC-3 cell growth when administered in combination with an oligonucleotide complementary to mRNA targeted to TGF-xcex1. Alone, the Her-1 oligonucleotide gave inhibition of cell growth equivalent to that achieved with an inverted (5xe2x80x2 to 3xe2x80x2) version of the same sequence.
Disclosed in U.S. Pat. No. 5,914,269 (Bennett, et al.) are oligonucleotides targeting Her-1 and methods of modulating the expression of Her-1 using said oligonucleotides. Three of these oligonucleotides and their analogs have also been used to investigate growth inhibition of SKOV3 cells, a human ovarian cancer cell line. In these studies it was demonstrated that all three oligonucleotides produced a significant reduction in Her-1 expression and protein production as well as in vitro growth inhibition of SKOV3 cells. Witters et al., Breast Cancer Research and Treatment, 1999, 53, 41-50.
Rearrangements or deletions of the Her-1 gene resulting in mutant Her-1 protein have been found in some cancers. The in-frame deletion from nucleotides 275-1075 in the Her-1 has been referred to as class I, Type I or Type III mutation. WO 96/16988 (Wong et al.) discloses cell lines capable of overexpressing Type III mutant Her-1, vaccines for inhibiting tumor formation comprising peptides similar to a fusion junction present in mutant human Her-1, antibodies raised against a cell line overexpressing Type III mutant Her-1, and oligonucleotides complementary to mRNA targeted to a Type III mutant Her-1 which decrease expression of a mutant Her-1. In a preferred embodiment, the oligonucleotide complementary to mRNA contains sequences from what were formerly distant portions of the normal Her-1 cDNA. The oligonucleotide must contain the sequence 5xe2x80x2-TACCTT-3xe2x80x2. An 18-mer oligonucleotide containing this sequence was found to downregulate mutant Her-1 levels when given at a 40 xcexcM dose in cultured cells which overexpressed Type III mutant Her-1.
The present invention provides new oligonucleotide compounds complementary to mRNA, as well as other oligonucleotide compounds, and compositions comprising the same together with methodologies for the use of certain compounds of the invention for interfering with translation of selected mRNA targets related to epidermal growth factor receptor.
The present invention is directed to oligonucleotides, especially those from 12-25 nucleotides in length, which are complementary to a nucleic acid molecule encoding Her-1, and which inhibit the expression of Her-1. In a preferred embodiment, the oligonucleotides comprise at least one phosphorothioate linkage. In other preferred embodiments, the oligonucleotides may comprise at least one 5-methyl cytosine, 2xe2x80x2-O-alkyl or 2xe2x80x2-fluoro modification. Pharmaceutical compositions comprising the oligonucleotides of the invention and a pharmaceutically acceptable carrier are also provided. Further provided are methods of modulating the expression of a human epidermal growth factor receptor in cells or tissues comprising contacting said cells or tissues with the oligonucleotide of the invention. These methods may be performed in vitro, in vivo or ex vivo. Methods of treating an animal having a hyperproliferative disease or condition by administering a therapeutically effective amount of an oligonucleotide of the invention for a time sufficient to ameliorate said hyperproliferative disease or condition are provided. The hyperproliferative disease or condition may be cancer, and in preferred embodiments the cancer is lung cancer, ovarian cancer, colon cancer or prostate cancer.
The present invention employs oligonucleotides for use in inhibiting the function of nucleic acid molecules encoding Her-1, ultimately modulating the amount of Her-1 produced. This is accomplished by providing oligonucleotides complementary to mRNA which specifically hybridize with mRNA or DNA encoding Her-1. Such hybridization with mRNA interferes with the normal role of mRNA and causes a modulation of its function. The functions of mRNA to be interfered with include all vital functions such as, for example, translocation of the RNA to the site of protein translation, translation of protein from the RNA, splicing of the RNA to yield one or more mRNA species, and catalytic activity which may be engaged in by the RNA. The overall effect of such interference with mRNA function is modulation of the expression of Her-1. In the context of this invention, xe2x80x9cmodulationxe2x80x9d means either an increase (stimulation) or a decrease (inhibition) in the expression of a gene. In the context of the present invention, inhibition is the preferred form of modulation of gene expression.
Oligonucleotides may comprise nucleotide sequences sufficient in identity and number to effect specific hybridization with a particular nucleic acid molecule. Such oligonucleotides are commonly described as xe2x80x9ccomplementary to mRNA.xe2x80x9d Oligonucleotides may also be directed to nucleotide sequences within the genome. Oligonucleotides are commonly used as research reagents and diagnostics. For example, oligonucleotides complementary to mRNA, which are able to inhibit gene expression with exquisite specificity, are often used by those of ordinary skill to elucidate the function of particular genes. Oligonucleotides complementary to mRNA are also used, for example, to distinguish between functions of various members of a biological pathway. This specific inhibitory effect has, therefore, been harnessed for research use.
The specificity and sensitivity of oligonucleotides is also harnessed by those of skill in the art for therapeutic uses. Oligonucleotides have been employed as therapeutic moieties in the treatment of disease states in animals and man. For example, workers in the field have now identified oligonucleotides complementary to mRNA, triplex oligonucleotides, and other oligonucleotide compositions which are capable of modulating expression of genes implicated in viral, fungal and metabolic diseases. Oligonucleotides complementary to mRNA have been safely administered to humans and numerous clinical trials are presently underway. It is thus established that oligonucleotides can be useful therapeutic modalities that can be configured to be useful in treatment regimes for treatment of cells, tissues and animals, especially humans.
It is preferred to target specific genes for attack by oligonucleotides complementary to mRNA. xe2x80x9cTargetingxe2x80x9d an oligonucleotide to a particular nucleic acid, in the context of this invention, is a multistep process. The process usually begins with the identification of a nucleic acid sequence whose function is to be modulated. This may be, for example, a cellular gene (or mRNA transcribed from the gene) whose expression is associated with a particular disorder or disease state, or a nucleic acid molecule from an infectious agent. In the present invention, the target is a nucleic acid molecule encoding Her-1. The targeting process also includes determination of a site or sites within this gene for the oligonucleotide interaction to occur such that the desired effect, e.g., detection or modulation of expression of the protein, will result. Within the context of the present invention, a preferred intragenic site is the region encompassing the translation initiation or termination codon of the open reading frame (ORF) of the gene. Since, as is known in the art, the translation initiation codon is typically 5xe2x80x2-AUG (in transcribed mRNA molecules; 5xe2x80x2-ATG in the corresponding DNA molecule), the translation initiation codon is also referred to as the xe2x80x9cAUG codon,xe2x80x9d the xe2x80x9cstart codonxe2x80x9d or the xe2x80x9cAUG start codon.xe2x80x9d A minority of genes have a translation initiation codon having the RNA sequence 5xe2x80x2-GUG, 5xe2x80x2-UUG or 5xe2x80x2-CUG, and 5xe2x80x2-AUA, 5xe2x80x2-ACG and 5xe2x80x2-CUG have been shown to function in vivo. Thus, the terms xe2x80x9ctranslation initiation codonxe2x80x9d and xe2x80x9cstart codonxe2x80x9d can encompass many codon sequences, even though the initiator amino acid in each instance is typically methionine (in eukaryotes) or formylmethionine (prokaryotes). It is also known in the art that eukaryotic and prokayotic genes may have two or more alternative start codons, any one of which may be preferentially utilized for translation initiation in a particular cell type or tissue, or under a particular set of conditions. In the context of the invention, xe2x80x9cstart codonxe2x80x9d and xe2x80x9ctranslation initiation codonxe2x80x9d refer to the codon or codons that are used in vivo to initiate translation of an mRNA molecule transcribed from a gene encoding Her-1, regardless of the sequence(s) of such codons. It is also known in the art that a translation termination codon (or xe2x80x9cstop codonxe2x80x9d) of a gene may have one of three sequences, i.e., 5xe2x80x2-UAA, 5xe2x80x2-UAG and 5xe2x80x2-UGA (the corresponding DNA sequences are 5xe2x80x2-TAA, 5xe2x80x2-TAG and 5xe2x80x2-TGA, respectively). The terms xe2x80x9cstart codon regionxe2x80x9d and xe2x80x9ctranslation initiation codon regionxe2x80x9d refer to a portion of such an mRNA or gene that encompasses from about 25 to about 50 contiguous nucleotides in either direction (i.e., 5xe2x80x2 or 3xe2x80x2) from a translation initiation codon. Similarly, the terms xe2x80x9cstop codon regionxe2x80x9d and xe2x80x9ctranslation termination codon regionxe2x80x9d refer to a portion of such an mRNA or gene that encompasses from about 25 to about 50 contiguous nucleotides in either direction (i.e., 5xe2x80x2 or 3xe2x80x2) from a translation termination codon. The open reading frame (ORF) or xe2x80x9ccoding region,xe2x80x9d which is known in the art to refer to the region between the translation initiation codon and the translation termination codon, is also a region which may be targeted effectively. Other target regions include the 5xe2x80x2 untranslated region (5xe2x80x2UTR), known in the art to refer to the portion of an mRNA in the 5xe2x80x2 direction from the translation initiation codon, and thus including nucleotides between the 5xe2x80x2 cap site and the translation initiation codon of an mRNA or corresponding nucleotides on the gene) and the 3xe2x80x2 untranslated region (3xe2x80x2UTR), known in the art to refer to the portion of an mRNA in the 3xe2x80x2 direction from the translation termination codon, and thus including nucleotides between the translation termination codon and 3xe2x80x2 end of an mRNA or corresponding nucleotides on the gene). mRNA splice sites may also be preferred target regions, and are particularly useful in situations where aberrant splicing is implicated in disease, or where an overproduction of a particular mRNA splice product is implicated in disease. Aberrant fusion junctions due to rearrangements or deletions are also preferred targets. Such rearrangements or deletions of the Her-1 gene resulting in mutant Her-1 protein are known to occur in some cancers. Once the target site has been identified, oligonucleotides are chosen which are sufficiently complementary to the target, i.e., hybridize sufficiently well and with sufficient specificity, to give the desired effect.
In the context of this invention, the term xe2x80x9coligonucleotidexe2x80x9d refers to an oligomer or polymer of ribonucleic acid or deoxyribonucleic acid. This term includes oligonucleotides composed of naturally-occurring nucleobases, sugars and covalent intersugar (backbone) linkages as well as oligonucleotides having non-naturally-occurring portions which function similarly. Such modified or substituted oligonucleotides are often preferred over native forms because of desirable properties such as, for example, enhanced cellular uptake, enhanced affinity for nucleic acid target and increased stability in the presence of nucleases. A discussion of antisense oligonucleotides and some desirable modifications can be found in De Mesmaeker et al., Acc. Chem. Res., 1995, 28, 366.
Specific examples of some preferred oligonucleotides envisioned for this invention include those containing modified backbones, for example, phosphorothioates, phosphotriesters, methyl phosphonates, short chain alkyl or cycloalkyl intersugar linkages or short chain heteroatomic or heterocyclic intersugar linkages. Most preferred are oligonucleotides with phosphorothioate backbones and those with heteroatom backbones, and in particular xe2x80x94CH2xe2x80x94NHxe2x80x94Oxe2x80x94CH2xe2x80x94, xe2x80x94CH2xe2x80x94N(CH3)xe2x80x94Oxe2x80x94CH2xe2x80x94 (known as a methylene (methylimino) or MMI backbone), xe2x80x94CH2xe2x80x94Oxe2x80x94N(CH3)xe2x80x94CH2xe2x80x94, xe2x80x94CH2xe2x80x94N(CH3)xe2x80x94N(CH3)xe2x80x94CH2xe2x80x94 and xe2x80x94Oxe2x80x94N(CH3)xe2x80x94CH2xe2x80x94CH2xe2x80x94 backbones, wherein the native phosphodiester backbone is represented as xe2x80x94Oxe2x80x94Pxe2x80x94Oxe2x80x94CH2xe2x80x94). The amide backbones disclosed by De Mesmaeker et al. (Id.) are also preferred. Also preferred are oligonucleotides having morpholino backbone structures, such as those described in Summerton and Weller, U.S. Pat. No. 5,034,506.
In other preferred embodiments, such as the peptide nucleic acid (PNA) backbone, the phosphodiester backbone of the oligonucleotide is replaced with a polyamide backbone, the nucleobases being bound directly or indirectly to the aza nitrogen atoms of the polyamide backbone. Nielsen et al., Science, 1991, 254, 1497.
Oligonucleotides may also contain one or more substituted sugar moieties. Preferred oligonucleotides comprise one of the following at the 2xe2x80x2 position: OH, SH, SCH3 F, OCN, OCH3OCH3, OCH3O(CH2)nCH3, O(CH2)nNH2 or O(CH2)nCH3 where n is from 1 to about 10; C1 to C10 lower alkyl, alkoxyalkoxy, substituted lower alkyl, alkaryl or aralkyl; Cl; Br; CN; CF3; OCF3; O-, S-, or N-alkyl; O-, S-, or N-alkenyl; SOCH3; SO2CH3; ONO2; NO2; N3; NH2; heterocycloalkyl; heterocycloalkaryl; aminoalkylamino; polyalkylamino; substituted silyl; an RNA cleaving group; a reporter group; an intercalator; a group for improving the pharmacokinetic properties of an oligonucleotide; or a group for improving the pharmacodynamic properties of an oligonucleotide, and other substituents having similar properties. A preferred modification includes 2xe2x80x2-methoxyethoxy (2xe2x80x2-Oxe2x80x94CH2CH2OCH3, also known as 2xe2x80x2-Oxe2x80x94(2-methoxyethyl)) (Martin et al., Helv. Chim. Acta, 1995, 78, 486). Other preferred modifications include 2xe2x80x2-methoxy (2xe2x80x2-Oxe2x80x94CH3), 2xe2x80x2-propoxy (2xe2x80x2-OCH2CH2CH3) and 2xe2x80x2-fluoro (2xe2x80x2-F). Similar modifications may also be made at other positions on the oligonucleotide, particularly the 3xe2x80x2 position of the sugar on the 3xe2x80x2 terminal nucleotide and the 5xe2x80x2 position of 5xe2x80x2 terminal nucleotide. Oligonucleotides may also have sugar mimetics such as cyclobutyls in place of the pentofuranosyl group.
Oligonucleotides may also include, additionally or alternatively, nucleobase (often referred to in the art simply as xe2x80x9cbasexe2x80x9d) modifications or substitutions. As used herein, xe2x80x9cunmodifiedxe2x80x9d or xe2x80x9cnaturalxe2x80x9d nucleobases include adenine (A), guanine (G), thymine (T), cytosine (C) and uracil (U). Modified nucleobases include nucleobases found only infrequently or transiently in natural nucleic acids, e.g., hypoxanthine, 6-methyladenine, 5-me pyrimidines, particularly 5-methylcytosine (also referred to as 5-methyl-2xe2x80x2 deoxycytosine and often referred to in the art as 5-me-C), 5-hydroxymethylcytosine (HMC), glycosyl HMC and gentobiosyl HMC, as well as synthetic nucleobases, e.g., 2-aminoadenine, 2-thiouracil, 2-thiothymine, 5-bromouracil, 5-hydroxymethyluracil, 8-azaguanine, 7-deazaguanine, N6 (6-aminohexyl)adenine and 2,6-diaminopurine. Kornberg, A., DNA Replication, W. H. Freeman and Co., San Francisco, 1980, pp.75-77 and Gebeyehu et al., Nuc. Acids Res., 1987, 15, 4513. A xe2x80x9cuniversalxe2x80x9d base known in the art, e.g., inosine, may be included. 5-me-C substitutions have been shown to increase nucleic acid duplex stability by 0.6-1.2EC (Sanghvi, Y. S., Crooke, S. T. and Lebleu, B., eds., Antisense Research and Applications, CRC Press, Boca Raton,1993, pp. 276-278) and are presently preferred base substitutions.
Another modification of the oligonucleotides of the invention involves chemically linking to the oligonucleotide one or more moieties or conjugates which enhance the activity or cellular uptake of the oligonucleotide. Such moieties include but are not limited to lipid moieties such as a cholesterol moiety, a cholesteryl moiety (Letsinger et al., Proc. Natl. Acad. Sci. USA, 1989, 86, 6553), cholic acid (Manoharan et al., Bioorg. Med. Chem. Let., 1994, 4, 1053), a thioether, e.g., hexyl-S-tritylthiol (Manoharan et al., Ann. N.Y. Acad. Sci., 1992, 660, 306; Manoharan et al., Bioorg. Med. Chem. Let., 1993, 3, 2765), a thiocholesterol (Oberhauser et al., Nucl. Acids Res., 1992, 20, 533), an aliphatic chain, e.g., dodecandiol or undecyl residues (Saison-Behmoaras et al., EMBO J., 1991, 10, 111; Kabanov et al., FEBS Lett., 1990, 259, 327; Svinarchuk et al., Biochimie, 1993, 75, 49), a phospholipid, e.g., di-hexadecyl-rac-glycerol or triethylammonium 1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate (Manoharan et al., Tetrahedron Lett., 1995, 36, 3651; Shea et al., Nucl. Acids Res., 1990, 18, 3777), a polyamine or a polyethylene glycol chain (Manoharan et al., Nucleosides and Nucleotides, 1995, 14, 969), or adamantane acetic acid (Manoharan et al., Tetrahedron Lett., 1995, 36, 3651), a palmityl moiety (Mishra et al., Biochim. Biophys. Acta, 1995, 1264, 229), or an octadecylamine or hexylamino-carbonyl-oxycholesterol moiety (Crooke et al., J. Pharmacol. Exp. Ther., 1996, 277, 923), all references being incorporated herein by reference. Oligonucleotides comprising lipophilic moieties, and methods for preparing such oligonucleotides are known in the art, for example, U.S. Pat. No. 5,138,045, U.S. Pat. No. 5,218,105 and U.S. Pat. No. 5,459,255, all of which are incorporated herein by reference.
The oligonucleotides of the invention may be provided as prodrugs, which comprise one or more moieties which are cleaved off, generally in the body, to yield an active oligonucleotide. One example of a prodrug approach is described by Imbach et al. in WO Publication 94/26764, incorporated herein by reference.
It is not necessary for all positions in a given oligonucleotide to be uniformly modified, and in fact more than one of the aforementioned modifications may be incorporated in a single oligonucleotide or even at a single nucleoside within an oligonucleotide. The present invention also includes oligonucleotides which are chimeric oligonucleotides. xe2x80x9cChimericxe2x80x9d oligonucleotides or xe2x80x9cchimeras,xe2x80x9d in the context of this invention, are oligonucleotides which contain two or more chemically distinct regions, each made up of at least one nucleotide. These oligonucleotides typically contain at least one region wherein the oligonucleotide is modified so as to confer upon the oligonucleotide increased resistance to nuclease degradation, increased cellular uptake, and/or increased binding affinity for the target nucleic acid. An additional region of the oligonucleotide may serve as a substrate for enzymes capable of cleaving RNA:DNA or RNA:RNA hybrids. By way of example, RNase H is a cellular endonuclease which cleaves the RNA strand of an RNA:DNA duplex. Activation of RNase H, therefore, results in cleavage of the RNA target, thereby greatly enhancing the efficiency of oligonucleotide inhibition of gene expression. Consequently, comparable results can often be obtained with shorter oligonucleotides when chimeric oligonucleotides are used, compared to phosphorothioate deoxyoligonucleotides hybridizing to the same target region. Cleavage of the RNA target can be routinely detected by gel electrophoresis and, if necessary, associated nucleic acid hybridization techniques known in the art.
The oligonucleotides in accordance with this invention preferably comprise from about 8 to about 30 nucleotides. It is more preferred that such oligonucleotides comprise from about 12 to 25 nucleotides. As is known in the art, a nucleotide is a base-sugar combination suitably bound to an adjacent nucleotide through a phosphodiester, phosphorothioate or other covalent linkage.
The oligonucleotides used in accordance with this invention may be conveniently and routinely made through the well-known technique of solid phase synthesis. Equipment for such synthesis is sold by several vendors including, for example, Applied Biosystems (Foster City, Calif.). Any other means for such synthesis known in the art may additionally or alternatively be employed. It is also known to use similar techniques to prepare other oligonucleotides such as the phosphorothioates and alkylated derivatives.
The oligonucleotides of the present invention can be utilized as diagnostics, therapeutics and as research reagents and kits. For therapeutics, an animal, preferably a human, suspected of having a disease or disorder which can be treated by modulating the expression of Her-1 is treated by administering oligonucleotides in accordance with this invention. The oligonucleotides of the invention can be utilized in pharmaceutical compositions by adding an effective amount of an oligonucleotide to a suitable pharmaceutically acceptable diluent or carrier. Use of the oligonucleotides and methods of the invention may also be useful prophylactically, e.g., to prevent or delay tumor formation.
The oligonucleotides of the present invention can be used as diagnostics for the presence of Her-1 -specific nucleic acids in a cell or tissue sample. For example, radiolabeled oligonucleotides can be prepared by 32p labeling at the 5xe2x80x2 end with polynucleotide kinase. Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, 1989, Volume 2, pg. 10.59. Radiolabeled oligonucleotides are then contacted with cell or tissue samples suspected of containing Her-1 mRNA (and thus, Her-1), and the samples are washed to remove unbound oligonucleotide. Radioactivity remaining in the sample indicates the presence of bound oligonucleotide, which in turn indicates the presence of nucleic acids complementary to the oligonucleotide, and can be quantitated using a scintillation counter or other routine means. Expression of nucleic acids encoding these proteins is thus detected.
Radiolabeled oligonucleotides of the present invention can also be used to perform autoradiography of tissues to determine the localization, distribution and quantitation of Her-1 for research, diagnostic or therapeutic purposes. In such studies, tissue sections are treated with radiolabeled oligonucleotide and washed as described above, then exposed to photographic emulsion according to routine autoradiography procedures. The emulsion, when developed, yields an image of silver grains over the regions expressing an Her-1 gene. Quantitation of the silver grains permits detection of the expression of mRNA molecules encoding Her-1 proteins and permits targeting of oligonucleotides to these areas.
Analogous assays for fluorescent detection of expression of Her-1 can be developed using oligonucleotides of the present invention which are conjugated with fluorescein or other fluorescent tags instead of radiolabeling. Such conjugations are routinely accomplished during solid phase synthesis using fluorescently-labeled amidites or controlled pore glass (CPG) columns. Fluorescein-labeled amidites and CPG are available from, e.g., Glen Research, Sterling, Va.
Oligonucleotides of the present invention directed to Her-1 can be used in diagnostics, therapeutics, prophylaxis, and as research reagents and kits. Because these oligonucleotides hybridize to nucleic acids encoding Her-1, sandwich and other assays can easily be constructed to exploit this fact. Hybridization of the oligonucleotides of the invention with a nucleic acid encoding an Her-1 can be detected by means known in the art. Such means may include conjugation of an enzyme to the oligonucleotide, radiolabelling of the oligonucleotide or any other suitable detection systems. Kits for detecting the presence or absence of Her-1 may also be prepared.
In the context of this invention, xe2x80x9chybridizationxe2x80x9d means hydrogen bonding, which may be Watson-Crick, Hoogsteen or reversed Hoogsteen hydrogen bonding, between complementary nucleotides. For example, adenine and thymine are complementary nucleobases which pair through the formation of hydrogen bonds. xe2x80x9cComplementary,xe2x80x9d as used herein, refers to the capacity for precise pairing between two nucleotides. For example, if a nucleotide at a certain position of an oligonucleotide is capable of hydrogen bonding with a nucleotide at the same position of a DNA or RNA molecule, then the oligonucleotide and the DNA or RNA are considered to be complementary to each other at that position. The oligonucleotide and the DNA or RNA are complementary to each other when a sufficient number of corresponding positions in each molecule are occupied by nucleotides which can hydrogen bond with each other. Thus, xe2x80x9cspecifically hybridizablexe2x80x9d and xe2x80x9ccomplementaryxe2x80x9d are terms which are used to indicate a sufficient degree of complementarity or precise pairing such that stable and specific binding occurs between the oligonucleotide and the DNA or RNA target. It is understood in the art that an oligonucleotide need not be 100% complementary to its target DNA sequence to be specifically hybridizable. An oligonucleotide is specifically hybridizable when binding of the oligonucleotide to the target DNA or RNA molecule interferes with the normal function of the target DNA or RNA to cause a loss of utility, and there is a sufficient degree of complementarity to avoid non-specific binding of the oligonucleotide to non-target sequences under conditions in which specific binding is desired, i.e., under physiological conditions in the case of in vivo assays or therapeutic treatment, or in the case of in vitro assays, under conditions in which the assays are performed.
The pharmaceutical compositions of the present invention may be administered in a number of ways depending upon whether local or systemic treatment is desired and upon the area to be treated. Administration may be topical (including ophthalmic, vaginal, rectal, intranasal, transdermal), oral or parenteral. Parenteral administration includes intravenous drip, subcutaneous, intraperitoneal or intramuscular injection, pulmonary administration, e.g., by inhalation or insufflation, or intrathecal or intraventricular administration. For oral administration, it has been found that oligonucleotides with at least one 2xe2x80x2-substituted ribonucleotide are particularly useful because of their absortion and distribution characteristics. U.S. Pat. No. 5,591,721 issued to Agrawal et al. Oligonucleotides with at least one 2xe2x80x2-methoxyethyl modification are believed to be particularly useful for oral administration. Formulations for topical administration may include transdermal patches, ointments, lotions, creams, gels, drops, suppositories, sprays, liquids and powders. Conventional pharmaceutical carriers, aqueous, powder or oily bases, thickeners and the like may be necessary or desirable. Coated condoms, gloves and the like may also be useful.
Compositions for oral administration include powders or granules, suspensions or solutions in water or non-aqueous media, capsules, sachets or tablets. Thickeners, flavoring agents, diluents, emulsifiers, dispersing aids or binders may be desirable.
Compositions for parenteral, intrathecal or intraventricular administration may include sterile aqueous solutions which may also contain buffers, diluents and other suitable additives.
The formulation of therapeutic compositions and their subsequent administration is believed to be within the skill of those in the art. Dosing is dependent on severity and responsiveness of the disease state to be treated, with the course of treatment lasting from several days to several months, or until a cure is effected or a diminution of the disease state is achieved. Optimal dosing schedules can be calculated from measurements of drug accumulation in the body of the patient. Persons of ordinary skill can easily determine optimum dosages, dosing methodologies and repetition rates. Optimum dosages may vary depending on the relative potency of individual oligonucleotides, and can generally be estimated based on EC50s found to be effective in vitro and in vivo animal models. In general, dosage is from 0.01 xcexcg to 100 g per kg of body weight, and may be given once or more daily, weekly, monthly or yearly, or even once every 2 to 20 years. Persons of ordinary skill in the art can easily estimate repetition rates for dosing based on measured residence times and concentrations of the drug in bodily fluids or tissues. Following successful treatment, it may be desirable to have the patient undergo maintenance therapy to prevent the recurrence of the disease state, wherein the oligonucleotide is administered in maintenance doses, ranging from 0.01 xcexcg to 100 g per kg of body weight, once or more daily, to once every 20 years.